PROJECT SUMMARY/ABSTRACT The mechanism of onset of neuroinflammation in fatal cALD in males with inherited X-linked adrenoleukodystrophy (X-ALD) disease remains unknown. 40% of male X-ALD patients develop fatal cerebral neuroinflammation (cALD) while remaining develop milder adrenomyeloneuropathy (AMN) characterized by axonopathy without neuroinflammation. The primary genetic defect in X-ALD (ABCD1 gene deletion) and the biochemical defect (accumulation of very long chain fatty acid; C>22:0 in plasma and tissues) cannot predict the onset of neuroinflammation in cALD. Our long-term goal is to dissect the molecular mechanism underlying differential phenotype development in X-ALD. The objective of this application is to identify integrated microRNA (miRNA) and metabolites that underlie the differential neuroinflammatory response in AMN and cALD human astrocytes. These astrocytes were differentiated from induced pluripotent stem cells (iPSCs), which in turn were generated by reprogramming of human control, AMN and cALD patient-derived untransformed fibroblasts. The neuroinflammatory response in X-ALD is likely initiated by astrocytes since the inflammatory areas in the X-ALD postmortem brain have cytokine secreting astrocytes but are devoid of activated microglia, T-cells and macrophages. Dysregulated miRNA and metabolite levels are associated with neuroinflammatory disease phenotype in a number of neurodegenerative diseases. Our preliminary proof-of-concept data, with next generation sequencing (miSeq) and untargeted metabolomics, identified miRNA and metabolites altered between healthy-control and cALD phenotype postmortem brain. Within the cALD brain white matter, miRNA and metabolite were altered between distant normal looking areas and neuroinflammatory areas adjacent to the plaque suggesting an association with disease progression. Our central hypothesis is that miRNA and metabolomic analysis in AMN and cALD human induced astrocytes will identify regulatory (miRNA) and active (metabolic) pathways that underlie the neuroinflammatory response and disease progression in cALD. To test our hypothesis we propose two specific aims: 1) To determine the miRNA altered in AMN and cALD astrocytes and 2) To identify metabolites altered between AMN and cALD astrocytes. This proposal is innovative, because it departs from the status quo by identifying for the first time, miRNA and metabolite pathways differentially regulating inflammatory response in human AMN and cALD astrocytes. In a step further, we will identify miRNA and metabolites reversed by CRISPR/Cas9 editing of AMN and cALD astrocytes with a functional copy of ABCD1. The proposed research is significant because the cellular mechanism(s) that lead to less severe AMN or neuroinflammatory cALD in response to the same ABCD1 mutation remain unknown even four decades after the identification of gene defect in X-ALD. As a result no therapy exists for AMN or cALD phenotypes. Impact: X-ALD was added to the federal newborn screening list in 2016, and with the rising rate of newly diagnosed cases there is urgent need to identify novel targets to develop effective therapies for AMN and cALD.